1. Field of the Invention
This invention relates to the use of covalently lipidated oligonucleotides comprising the CpG dinucleotide unit, or an analogue thereof, as immunostimulatory agents. Lipidated oligonucleotides with special backbones, lipidated oligonucleotides with fewer than eight nucleotides, and lipidated oligonucleotides comprising a plurality of CpG dinucleotide-containing segments connected by a long internucleoside linkage are of particular interest.
2. Description of the Background Art
Effective host defense against invading microorganisms requires the detection of foreign pathogens and the rapid deployment of an antimicrobial effector response (Krutzik et al, 2001). Indeed, the innate immune system detects the presence and the nature of the infection by recognizing the constitutive and conserved products of microbial metabolism, which can be viewed as molecular signatures of microbial invaders (Janeway, 1992), and also called pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by various pattern-recognition receptors (PRRs) of the innate immune system, which are expressed on the cell surface, in intracellular compartments, or secreted into the blood stream and tissue fluids. Also, the innate immune system provides the first line of host defense, and controls the initiation and determination of the effector class of the adaptive immune response (Medzhitov, 2001).
Toll-Like Receptor Ligands
The recent discovery and characterization of the Toll-like receptor (TLR) family have incited new interest in the field of innate immunity. TLRs are also pattern-recognition receptors (PRRs) that have a unique and essential function in animal immunity. In mammalian species there are at least ten TLRs currently known (Medzhitov, 2001), and each seems to have a distinct function in innate immune recognition. Dozens of TLR ligands have been identified (Akira et al, 2001).
Though quite diverse in structure and origin, TLR ligands have several common features. For instance, most TLR ligands are conserved microbial products (PAMPs) that signal the presence of infection; and many individual TLRs can recognize several structurally unrelated ligands. It is obvious that TLR ligands are potent activators of innate immune system, which in turn directs and determines the adaptive immune response.
CpG Motifs
Probably the most enigmatic example of pattern recognition is the recognition of unmethylated CpG motifs in bacterial DNA by TLR9 (Hemmi et al, 2000). As a matter of fact, unmethylated bacterial DNA in a particular sequence context (the so-called CpG motif) has been known for its potent immune stimulatory activity for quite some time (Krieg et al, Nature, 1995).
WO98/18810 (University of Iowa Research Foundation) teaches that certain nucleic acids containing unmethylated CpG dinucleotides activate lymphocytes in a subject and redirect a subject's immune responses from a Th2 to a TH1, i.e., increase production of Th1 cytokines including IL-12, IFN-gamma and GM-CSF. In particular, it discloses an isolated immunostimulatory nucleic acid sequence of about 8-30 bases “represented” by the formula5′ N1X1CGX2N2 3′where at least one nucleotide separates consecutive CpGs; X1 is A, G or T, X2 is C or T, N is any nucleotide, and N1+N2 is 0-26 nucleotides, with the proviso that the latter does not contain a CCGG quadmer or more than one CCG or CGG trimer. With respect to stimulation of murine cells, a preference is expressed for a CpG flanked by two 5′ purines (preferably GpA) and two 3′ pyrimidines (preferably TpT or TpC).
The authors reported that oligomers shorter than 8 bases were non-stimulatory (page 25, lines 16-17); the tested oligomer was a 7-mer (Table 1, 4e). See also Sonehara, et al., “Hexamer Palindromic Oligonucleotides with 5′-CG-3′ Motif(s) Induce Production of Interferon,” J. Interferon & Cytokine Res., 16:799-803 (1996)(IFN-inducing activity of ACGT “insignificant”). In contrast, in the present invention, we have found that if lipidated, even a dinucleotide by itself has activity.
WO98/40100 (Ottawa Civic Loeb Research Laboratory, Qi-Agen GmbH, and University of Iowa Research Foundation), WO99/51259 (University of Iowa Research Foundation), WO99/61056 (Loeb Health Research Institute at the Ottawa Hospital, CPG Immunopharmaceuticals, Inc.) have similar teachings. While WO98/40100 defines an oligonucleotide as being at least five bases in length, it nonetheless teaches that for the desired immunostimulatory activity, at least 8 bases are needed. WO01/97843 says that the immunostimulatory nucleic acid can have any length greater than 6 nucleotides (p. 7, l. 12). WO99/61056 (p3, L30) implies that a hexanucleotide can induce mucosal immunity, although an octanucleotide is preferred (p 8, l. 17).
WO01/12804 (Hybridon, Inc.) teaches that it is desirable that the two bases immediately flanking the CpG on its 5′ end be 2′-OMe substituted.
WO01/97843 (University of Iowa Research Foundation) teaches that it is desirable that the CpG oligonucleotide be “T-rich”, i.e., greater than 25% T, more preferably, greater than 40%, 50%, 60%, 80% or 90% T. It teaches that it is desirable that it comprise at least one poly-T motif consisting of at least three consecutive T bases. It expresses a preference for longer poly-T motifs (at least 4-9 Ts) and for a plurality of poly-T motifs (at least 2-8). Likewise, it discloses the desirability of poly-A, poly-C, and poly-G motifs.
WO00/54803 (Panacea Pharmaceuticals, LLC) relates to use of the CpG-containing oligonucleototides to ameliorate allergic responses to immunogens. See also DeKruyff, U.S. Pat. No. 6,086,898 (2000), on converting a Th2-type allergic immune response into a Th1-type immune response.
WO01/07055 teaches use of a CpG-oligonucleotide having a particular topology, for higher stability in vivo.
Lipoteichoic Acid; Doubse-Stranded Ribonucleic Acid
Structurally related lipo-teichoic acid (LTA) from Gram-positive bacteria and double-stranded RNA (dsRNA) from viruses are also well known for their properties of activating host innate immunity. Recent studies have shown that TLR4 is involved in the recognition of LTA (Takeuchi et al, 1999) and TLR3 functions as a cell-surface receptor for dsRNA (Alexopoulou et al, 2001).
Teichoic acids are polyol phosphate polymers, with either ribitol or glycerol linked by phosphodiester bonds. Substituent groups on the polyol chains of the naturally occurring teichoic acids can include D-alanine (ester-linked), N-acetylglucosamine, and glucose. In the ribitol teichoic acids, there are 1,5-phosphodiester linkages. In the glycerol teichoic acids, there are 1,2- or 1,3-phosphodiester linkages. The glycerol teichoic acids may be unsubstituted, or substituted (e.g. alanyl or glycosyl) at the remaining position.
Glycerol Nucleic Acids
Usman, Juby and Ogilvie, “Preparation of Glyceronucleoside Phosphoramidite Synthons and Their Use in the Sold Phase Synthesis of Acyclic Oligonucleotides” Tetrahedron Lett., 29: 4831-4 (1988) descibes the synthesis of homooligomers (2-8 units long) of the nucleotides in which either adenine or thymine are part of a glyceronucleoside. It is noted in passing that glyceronucleosides per se, especially those containing purines (adenine and thymine) as the base component, are potent antiviral agents.
Schneider and Benner, “Oligonucleotides Containing Flexible Nucleoside Analogues,” J. Am. Chem. Soc. 112: 453-55 (1990) disclose oligonucleotides in which ribose is replaced by a glycerol derivative. However, they found that the change in backbone reduced the ability of the oligonucleotide to form a stable duplex structure. Each glycerol nucleoside reduced the melting point of the duplex DNA by about 9-15° C. The oligonucleotides synthesized by Schneider and Benner were at least 9 bases long, and none of them comprised 5′-CG-3′.
Other Oligonucleotide Analogues
Oligonucleotide analogues, especially those with modified internucleoside linkages, are known in the art. See e.g. Cook, U.S. Pat. No. 5,717,083; Weis, U.S. Pat. No. 5,677,439; Rosch, U.S. Pat. No. 5,750,669; Cook, U.S. Pat. No. 6,114,513; Cook, U.S. Pat. No. 6,111,085; Uhlmann, et al., “PNA: Synthetic Polyamide Nucleic Acids with Unusual Binding Properties,” Angew. Chem. Int. Ed. 37:2796-2823 (1998).
Chemically Modified Nucleic Acids
Englisch and Gauss, “Chemically Modified Oligonucleotides as Probes and Inhibitors”, Angew. Chem. Int. Ed. Engl., 30: 613-29 (1991) make reference to oligonucleotides modified to include psoralen, acridine, biotin, or enzymes.
Known terminal radicals (hydroxyl substituents) include 1-ethoxyethyl, ethoxymethyl, benzhydryl, benzyl, trityl, monomethoxytrityl, dimethoxytrityl, methyl, ethyl, acetyl, tosyl, tetrahydropyranyl, trifluoroacetyl, aminoacyl, glycyl, leucyl, cyanoethyl, anisyl, benzyl, and phenyl, and, as bifunctional protecting groups (usually bridging 2′ and 31),
Known terminal glycol-protecting (bifunctional) radicals, bridging the 2′ and 3′ hydroxyls unless otherwise indicated, include isopropylidene, borate, and carbonyl 2′:3′-phosphate (cyclic).
N-protecting radicals used in synthetic work include benzoyl, benzyl, tosyl, trityl, anisoyl, benzhydryl (diphenylmethyl), monomethoxytrityl (p-anisyldiphenylmethyl), dimethoxytrityl (di-p-anisylphenylmethyl), tetrahydropyranyl, dansyl, and N-cyclohexyl-N′[-(4-methylmorpholino)amidino].
Phosphoric acid protecting groups include 5′-cyanoethyl; 3′(or 2′)-cyanoethyl, anisyl (MeOPh), benzyl, and phenyl.
Lipidated Nucleic Acids
Hostetler, U.S. Pat. No. 5,827,831 teaches that oral delivery of many clases of drugs is facilitated by converting drugs having suitable functional groups to 1-O-alkyl, 1-O-acyl, 1-S-acyl, and 1-S-alkyl-sn-glycero-3-phosphate derivatives; he refers to these derivatives as lipid prodrugs. The classes of drugs taught by Hostetler include “anticancer agents, comprising nucleoside analogues, for example, 9-beta-D-arabinofuranosylcytosine (hereinafter, cytosine arabinoside or ara-C), 9-beta-D-arabinofuranosyladenine (hereinafter, adenine arabinoside or ara-A), 5-fluorouridine, 6-mercaptopurine riboside, or 2′-ara-fluoro-2-chlorodeoxyadenosine”, and “antiviral nucleosides, particularly the 1-O-alkyl phospholipid derivatives of those antiviral nucleosides disclosed in U.S. Pat. No. 5,223,263”. In the antiviral category, specific reference is made to 3′deoxy, 3′-azidothymidine (AZT), acyclovir, foscarnet, ganciclovir, idoxuridine, ribavarin, 5-fluoro-3′-thia-2′,3′-dideoxycytidine, trifluridine, and vidarabine. There is no reference to lipidation of any oligonucleotides.
Sridhar, U.S. Pat. No. 5,756,352 discloses thiocationic lipid-nucleic acid conjugates. The cationic lipid binds noncovalently to the anionic nucleic acid molecules. The present invention requires covalent attachment of a lipophilic group to the oligonucleotide.
Cheng, et al. U.S. Pat. No. 5,646,126 discloses double stranded oligonucleotides having a lipophilic group, preferably a steroid structure, attached to the 3′ end. The oligonucleotides comprised 8-18 bases (per strand). They do not disclose or suggest any shorter oligonucleotides, or any molecules without at least some double-stranded structure.
Cheng et al. conceived of three types of molecules with double stranded structure. In type 1, the oligonucleotide is palindromic, so two molecules together form a duplex. In type 2, there are two different but substantially complementary strands, which hybridize to form the duplex. Finally, in type 3, the oligonucleotides are at least partially self-complementary, so they “hairpin” to form a double-stranded structure.
The oligonucleotides of interest to them were those with anticancer activity. They do not disclose or suggest that any of his oligonucleotides have immunostimulatory activity.
Several of these oligonucleotides (e.g., 120 H, 128 H, 001 H, 167 H, 002 H, 089 H, 589 H, 178 H, 678 H) comprise 5′-CG-3′. The 3′ modifications of sequence 128H (CACACGTGTG)(SEQ ID NO: 1) included cholesterol*, hexylamine, acridine, hexanol, hexadecane, cholestanol*, ergosterol, stigmastanol*, stigmasterol*, and methyl-lithacholic acid; only the starred modifications had anticancer activity (see Cheng FIG. 10). Thus, Cheng et al. discouraged further experimentation with 3′ lipophilic modifications other than those with the steroid skeleton of his Formula 1. Cheng et al. do not disclose or suggest any 5′ lipophilic attachments.
While Cheng et al. contemplated the possibility of backbone modification, especially, phosphorothioate (P-S0 linkages, they did not specifically suggest peptide-nucleic acid (PNA) or glycerol nucleic acid (GNA) backbones. Indeed, since GNA backbones reduce duplex stability, use of a GNA backbone would have been contrary to Cheng et al.'s teaching of oligonucleotide duplexes.
Targeted Nucleic Acids
Manoharan, U.S. Pat. No. 6,300,319 discloses attaching a cell surface receptor ligand to an oligonucleotide to facilitate delivery of the oligonucleotide to the cell in question. Manoharan notes that natural oligonucleotides are polyanionic and poorly penetrate cells, while the methylphosphonates are neutral and are taken up much more readily. The ligands contemplated by Manoharan are primarily carbohydrates (targeting cell surface lectins) sch as galactose, N-acetylgalactosamine, fucose, mannose, and sialic acid. These would not be considered lipophilic groups (see Table K-2, below).